7. REQUERIMIENTOS TÉCNICOS DE CONEXIÓN
7.1.2. Cumplimiento de los P.O y de las condiciones técnicas de conexión
First it was investigated whether the modified CPs would still assemble into VLPs in response to pH changes under conditions where the ELP domain would not undergo its phase transition. To induce assembly, the CPs were dialyzed against a pH 5.0 acetate buffer and visualized by transmission electron microscopy (TEM). The ELP-CPs formed VLPs with a diameter of 28 nm (Figure 5.2 left). Dynamic light scattering (DLS) showed that these assembly conditions yielded a uniform assembly product with an estimated diameter of 31 nm (Figure 5.3a), with minimal aggregation. Size exclusion chromatography
Designing two self-assembly mechanisms into one viral capsid protein
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(SEC) inline with multi-angle laser light scattering (MALLS) indicated that the VLPs, which eluted at 10.2 mL on a Superose 6 10/300 GL column (Figure 5.4a), had a molecular weight of 4.0 MDa, in line with 180 CPs of 22253.4 Da (theoretically 4.0 MDa).
These results are consistent with VLPs resembling native-like T = 3 particles. The
assembly at pH 5.0 was of high affinity, as no peak corresponding to dimers (expected at 17.0 mL; Figure 5.4a) was observed, and had high fidelity, as no other aggregates or assembly products were observed. Asymmetric-flow field-flow fractionation (AF4) coupled to MALLS was used to further support the results observed by SEC-MALLS (Figure 5.4b). AF4 also showed a highly effective assembly process and a molecular weight
that corresponded to T = 3 VLPs. In comparison, assembly of WT-CP under similar
conditions occurred with lower efficiency and always left some residual dimer.[32]
Figure 5.2. Uranyl acetate-stained TEM micrographs of CPs assembled into VLPs. Overviews (top) and enlarged
Chapter 5
To ascertain the particle geometry and understand the disposition of the ELP blocks, we performed cryogenic electron microscopy (cryo-EM) and three-dimensional reconstruction of the pH-induced H6-ELP-[V4L4G1-9]-CP(ΔN26) VLPs (Figure 5.5a and
b). It was observed that the VLPs consisted of two shells (Figure 5.5c and e). As the morphology of the outer shell was identical to that of the empty T = 3 CCMV capsid,[19, 33]
the electron density of the inner shell could only be explained by the presence of the ELPs, indicating that the ELP domain indeed was in the interior of the VLPs. Moreover, it tentatively appeared that in this environment the ELPs self-assembled because of their high local concentration.
Figure 5.3. DLS measurements of SEC-purified assembled H6-ELP-[V4L4G1-9]-CP(ΔN26) under (a) pH-induced
conditions and (b) ELP-induced conditions. Three measurements were performed for each sample.
Figure 5.4. (a) SEC-MALLS chromatograms of H6-ELP-[V4L4G1-9]-CP(ΔN26). The solid lines show the normalized
differential refractive index signals, and the dotted lines show the molecular masses of the complexes. (b) AF4- MALLS chromatograms of H6-ELP-[V4L4G1-9]-CP(ΔN26). The solid lines show the normalized absorbance at 280
nm, and the dotted lines show the molecular masses of the complexes. It is noteworthy that the larger VLPs elute in front of the CP dimers by SEC, whereas, as expected, this elution behavior is reversed in case of AF4.
Designing two self-assembly mechanisms into one viral capsid protein
123 5.2.3. ELP-induced assembly of virus-like particles
After it was established that the ELP-CPs assembled into T = 3 VLPs under pH-induced
conditions, ELP-induced assembly was assayed. The ELP Tt can be tuned by several
factors, such as molecular weight, amino acid composition and salt and protein concentration. As the polymer length and the amino acids at the fourth positions in the pentapeptide repeat were already determined by the design of the polypeptide, the salt concentration was used to adjust Tt into a range suitable for the CCMV CP. Increasing the
NaCl concentration to 2.5 M lowered the Tt of the ELP of the fusion protein to below
room temperature. Therefore, to induce assembly, ELP-CP dimers were dialyzed against a pH 7.5 buffer containing 2.5 M NaCl, and then the samples were analyzed by TEM. Similar to the pH-induced assembly, we again observed assembly of the ELP-modified capsid proteins into monodisperse VLPs, but these had a diameter of 18 nm (Figure 5.2 right). These assemblies were then analyzed by DLS, and a diameter of 25 nm was estimated (Figure 5.3b). These smaller VLPs were also analyzed by SEC-MALLS and AF4-MALLS (Figure 5.4a and b). Triggering the stimulus response of the ELPs led to assembly of the dimers into 1.3 MDa VLPs with no residual dimers, indicating again high assembly efficiency. This mass corresponds to 60 CPs of 22253.4 Da, suggesting a T = 1
Chapter 5
Figure 5.5. Cryo-EM micrographs and reconstructions of H6-ELP-[V4L4G1-9]-CP(ΔN26) VLPs. Selected images of the
frozen-hydrated specimens of the (a) pH-induced VLPs and (b) ELP-induced VLPs. The inserts show the translationally-aligned averaged images. Note that both VLPs appear to have the morphology of two concentric shells. Cryo-EM reconstructions of VLPs viewed along the icosahedral twofold axis: (c, e) pH-induced assembly yielded 90-dimer T = 3 particles; (d, f) ELP-induced assembly yielded 30-dimer T = 1 particles. The top panels (c, d) depict surface views, and the bottom panels (e, f) present equatorial views to show the interior. Ovals, triangles, and pentagons indicate locations of twofold, threefold, and fivefold axes, respectively.
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Cryo-EM and image reconstruction were employed to verify that the ELP-induced assembly yielded T = 1 icosahedra (Figure 5.5d and f). Again, two shells surrounding a
small unoccupied inner volume were observed. The outer shell showed the pentameric turrets expected for CCMV,[19] and the electron density of the inner shell was again
attributable to ELP. In our construct, the ELP domain was designed to interact with neighboring ELP domains. This could explain the tighter packing that resulted in smaller VLPs after ELP-induced assembly. This hypothesis was corroborated by previous reports in the literature showing that CPs lacking both the positively charged RNA-binding
domain and the β-hexamer (residues 3-36) could be assembled into mainly pseudo T = 2
and T = 1 and a small fraction of T = 3 icosahedrons in a pH-dependent manner.[19]
Formation of T = 1 particles was also observed for truncated CPs lacking the N-terminal
region and for full length WT-CPs via kinetic trapping upon addition of short oligonucleotides or anionic polymers.[32, 34-36] However, the quantitative formation of T =
1 particles reported here is unprecedented.
A NaCl concentration of 2.5 M was applied initially to ensure that all of the ELP-CPs were assembled. Next, a series of NaCl concentrations was used to investigate the minimum concentration required to obtain assembly at room temperature and pH 7.5. It was found that a concentration of 1.8 M NaCl in the pH 7.5 buffer was enough to induce full assembly of the ELP-CPs (Figure 5.6 and Figure 5.7).
Figure 5.6. Size exclusion chromatograms for assembly of H6-ELP-[V4L4G1-9]-CP(ΔN26) in pH7.5 buffer with
varying NaCl concentrations. The assembly was analyzed in pH 7.5 buffer with 1.4 M NaCl (red line), 1.5 M NaCl (blue line) and 1.6 M NaCl (black line) at 20 °C.
Chapter 5
To demonstrate that the ELP was responsible for the assembly at high salt concentrations, we investigated whether CPs lacking the stimulus-responsive peptide could self-assemble under similar conditions. WT-CP and H6CP[21] were dialyzed into pH
7.5 buffer with 1.8 M NaCl. However, under these conditions, the control proteins were detectable only as dimers (Figure 5.7).
At NaCl concentrations below 1.8 M, only a fraction of the ELP-CPs assembled, resulting in a bimodal distribution of capsid and dimer consistent with virus assembly
theory (Figure 5.6).[37] This is notable because when free ELP undergoes a phase
transition, it leads to complete aggregation. This difference is probably a result of the fact that the ELPs are sequestered on the inside of the VLPs, thus limiting the number of ELPs that can participate in a given nucleation event. To verify that this was the reason for incomplete assembly, a sample was diluted twice and measured again by SEC after overnight incubation. As expected, the VLP:dimer ratio changed from 0.39:1 to 0.17:1 (based on the areas under the peaks; Figure 5.8). This showed that relatively more ELP- CP is present as dimers at lower protein concentrations and that the assembly is reversible. In the previous experiments, the assembly of ELP-CP was induced by starting from CP dimers and applying either the ELP or the pH-based assembly trigger. When both pathways were triggered simultaneously (pH 5.0 with 2.5 M NaCl), the ELP-CP assembled into T = 1 VLPs (Figure 5.9a). Interestingly, sequential triggering of the stimuli resulted in
detection of the product of the first trigger; i.e. pH-induced, followed by ELP-induced
assembly led to T = 3 particles, whereas ELP-induced, followed by pH-induced particle
formation yielded T = 1 particles. When pH-induced T = 3 VLPs were exposed to a
simultaneous increase of pH and NaCl concentration a mixture of T = 1 and T = 3
icosahedra was obtained. Potentially, longer incubation could result in complete transition towards T = 1 particles. Notably, immediate switching from conditions for ELP-induced
VLPs to pH-induced conditions resulted in precipitation. The diagram in Figure 5.9b summarizes the observed assembly products under various conditions.
Designing two self-assembly mechanisms into one viral capsid protein
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Figure 5.7. Size exclusion chromatograms for assembly of control proteins and SDS-PAGE of the CPs. (a) The
assembly of H6-ELP-[V4L4G1-9]-CP(ΔN26) (blue solid line), H6CP (red solid line) and WT-CP (black dashed line) in a
pH 7.5 buffer with 1.8 M NaCl. (b) Coomassie-stained SDS-PAGE depicts the CPs used in this study: H6-ELP-
[V4L4G1-9]-CP(ΔN26) (lane 1), H6CP (lane 2) and WT-CP (lane 3).
Figure 5.8. SEC chromatogram for self-assembly at two different concentrations of protein. The assembly of H6-
ELP-[V4L4G1-9]-CP(ΔN26) at 1.0 mg/mL was analyzed in pH 7.5 buffer with 1.3 M NaCl (black line) and after
dilution of the sample to 0.5 mg/mL and overnight incubation (red line). The areas under the peak were determined to calculate the ratio between the VLPs and the dimers.
Chapter 5
Figure 5.9. (a) SEC-MALLS chromatograms of H6-ELP-[V4L4G1-9]-CP(ΔN26). The solid lines show the differential
refractive index signals, and the dotted lines show the molecular masses of the complexes. (b) H6-ELP-[V4L4G1-9]-
CP(ΔN26) diagram for assembly under various conditions.
Finally, the temperature-responsive assembly of ELP-CPs was examined. As concluded from the previous experiments, only a minor fraction of ELP-CPs was assembled at room temperature in the 1.3 M NaCl buffer at pH 7.5. Therefore, a sample of ELP-CPs under these conditions was incubated at 35 °C for 15 min and then again analyzed by SEC at this temperature. The higher temperature resulted in efficient assembly of the dimers into T = 1 particles (Figure 5.10). From this experiment it can also be
concluded that the assembly is a rather fast process, as it was completed within 15 min.
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Temperature-responsive assembly was also confirmed by TEM. TEM grids were prepared of ELP-CP at room temperature in pH7.5 buffer with 2.0 M and 2.3 M NaCl (Figure 5.11a and c, respectively). Next, the CP solutions were cooled and incubated for 30 min at 4 °C and then again TEM grids were prepared (Figure 5.11b and d). TEM analysis at 20 °C for ELP-CP in pH 7.5 with 2.0 M NaCl showed VLPs with a diameter of 18 nm, whereas these disappeared after cooling to 4 °C. In the pH 7.5 buffer with 2.3 M NaCl, no temperature effect was observed between 4 °C and 20 °C, and in both cases VLPs with a diameter of 18 nm were detected, probably because the salt concentration decreased the Tt
to below 4 °C.
Figure 5.11. Uranyl acetate-stained TEM micrographs of H6-ELP-[V4L4G1-9]-CP(ΔN26) prepared at different
temperatures and NaCl concentrations. The assembly was analyzed in pH 7.5 buffer: (a) 2.0 M NaCl at 4 °C; (b) 2.0 M NaCl at 20 °C; (c) 2.3 M NaCl at 4 °C; (d) 2.3 M NaCl at 20 °C.
5.3. Conclusion
This work shows the feasibility of combining two structural protein elements to generate a system with emergent properties. We constructed a capsid protein fusion that can be self- assembled via two mechanisms into two different, well-defined structures. To construct such a system, thermally responsive ELPs were genetically combined with CCMV CPs. Under pH-induced assembly conditions, the modified CPs assembled into 28 nm diameter VLPs with a T = 3 icosahedral architecture. Thermally responsive ELP-induced assembly
resulted in the efficient production of 18 nm diameter T = 1 icosahedra. This architecture
Chapter 5
the assembly was induced by triggering the phase transition of ELP, but the architecture was controlled by the CPs. The present system could find application in switchable encapsulation of enzymes to control their activity. The VLPs could also be used as a capture and release system for therapeutics. The confined internal volume with a relatively high ELP concentration could also lead to a better understanding of ELP structural characteristics.
5.4. Acknowledgements
Inge Minten is gratefully acknowledged for her contribution to this chapter. We thank Joseph Wang, Chenglei Li and Adam Zlotnick for the fruitful collaboration and the useful discussions. Jeroen Cornelissen and Roeland Nolte are thanked for valuable discussions.
5.5. Experimental section
Materials
Restriction enzymes, ligase and kinase were obtained from New England Biolabs. The DNA oligos were synthesized by Biolegio (Nijmegen, The Netherlands). The wild-type capsid protein (WT-CP) was provided by J.J.L.M. Cornelissen from University of Twente, The Netherlands. Ampicillin and chloroamphenicol were purchased from MP Biomedicals. Lysozyme and isopropyl β-D-1- thiogalactopyranoside (IPTG) were obtained from Sigma-Aldrich.
Two types of buffers were used, a pH 5.0 buffer (50 mM NaOAc, 500 mM NaCl, 10 mM MgCl2, 1 mM
EDTA, pH 5.0 set with HCl) and a pH 7.5 buffer (50 mM TrisHCl, 500-2500 mM NaCl, 10 mM MgCl2,
1 mM EDTA, pH 7.5 set with HCl). The NaCl concentration in the latter buffer was varied between 500 and 2500 mM. Prior to use, all buffers were filtered over a Millipore 0.2 µm filter and for the SEC- MALLS analysis, the buffers were also degassed.
Elastin-like polypeptide nomenclature
ELP constructs are usually described using the notation ELP[XiYjZk-n], where the capital letters between
the brackets indicate the single letter amino acid code for the residues at the Xaa position in the pentapeptide Val-Pro-Gly-Xaa-Gly. The subscript stands for the ratio of the guest residues and the n
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131
Construction of expression vector
The pET-15b-H6-CP vector coding for bacterial expression of the histidine-tagged CCMV capsid protein
was previously constructed as described by Minten et. al.[21] For the insertion of the ELP and the removal
of the RNA-binding domain, two sets of DNA oligos with a complementary overhang were designed (see Table 5.1). These two sets of oligos were annealed and the resulting insert encoded for a histidine-tag, the ELP[V4L4G1-9] and a short fragment of the CP with a 5’ NcoI and a 3’ AgeI restriction site. First, the
pET-15b-H6-CP vector was digested with NcoI-HF™ and AgeI-HF™, and then the product was
purified by agarose gel electrophoresis. Subsequently, the annealed inserts were ligated into the digested vector. The linear product was again purified by agarose gel electrophoresis and then it was phosphorylated and ligated to yield the pET-15b-H6-ELP[V4L4G1-9]-CP(ΔN26). This plasmid was
transformed into E. coli XL1-Blue cells, the DNA was extracted and the sequence was confirmed by
DNA sequencing. The plasmid was transformed into E. coli BLR(DE3)pLysS cells (Novagen, MERCK),
which were used for the expression of H6-ELP[V4L4G1-9]-CP(ΔN26).
Table 5.1. DNA sequence of oligos used for construction of expression vector
Name Sequence
Forward-1 5'- CATGGGCCATCATCATCATCATCACGTTCCGGGCGTCGGTGTTCCTGGATTAGGTGTTCC … …
Reverse-1 3'- CCGGTAGTAGTAGTAGTAGTGCAAGGCCCGCAGCCACAAGGACCTAATCCACAAGG … …
(continued) Forward-1 … … AGGTGTAGGTGTTCCAGGACTCGGTGTTCCTGGTGTAGGTGTTCCTGGTTTAGGTG -3'
(continued) Reverse-1 … … TCCACATCCACAAGGTCCTGAGCCACAAGGACCACATCCACAAGGACCAAATCCACAAGG -5'
Forward-2 5'- TTCCAGGTGGCGGTGTTCCTGGTGTGGGAGTTCCTGGTTTAGGTCTCGAGGTGGTCCAAC … …
Reverse-2 3'- TCCACCGCCACAAGGACCACACCCTCAAGGACCAAATCCAGAGCTCCACCAGGTTG … …
(continued) Forward-2 … … CTGTTATTGTAGAACCCATCGCTTCAGGCCAAGGCAAGGCTATTAAAGCATGGA -3'
(continued) Reverse-2 … … GACAATAACATCTTGGGTAGCGAAGTCCGGTTCCGTTCCGATAATTTCGTACCTGGCC -5'
Protein sequences WT-CP MSTVGTGKLTRAQRRAAARKNKRNTRVVQPVIVEPIASGQGKAIKAWTGYSVSKWTASCAA AEAKVTSAITISLPNELSSERNKQLKVGRVLLWLGLLPSVSGTVKSCVTETQTTAAASFQV ALAVADNSKDVVAAMYPEAFKGITLEQLTADLTIYLYSSAALTEGDVIVHLEVEHVRPTFD DSFTPVY H6-CP GSSHHHHHHSSGLVPRGSHMMSTVGTGKLTRAQRRAAARKNKRNTRVVQPVIVEPIASGQG KAIKAWTGYSVSKWTASCAAAEAKVTSAITISLPNELSSERNKQLKVGRVLLWLGLLPSVS GTVKSCVTETQTTAAASFQVALAVADNSKDVVAAMYPEAFKGITLEQLTADLTIYLYSSAA LTEGDVIVHLEVEHVRPTFDDSFTPVY
Chapter 5 H6-ELP[V4L4G1-9]-CP(ΔN26) GHHHHHHVPGVGVPGLGVPGVGVPGLGVPGVGVPGLGVPGGGVPGVGVPGLGLEVVQPVIV EPIASGQGKAIKAWTGYSVSKWTASCAAAEAKVTSAITISLPNELSSERNKQLKVGRVLLW LGLLPSVSGTVKSCVTETQTTAAASFQVALAVADNSKDVVAAMYPEAFKGITLEQLTADLT IYLYSSAALTEGDVIVHLEVEHVRPTFDDSFTPVY
Expression and purification of capsid proteins
For a typical expression, 100 mL 2xYT medium, supplemented with ampicillin (100 mg/L) and chloroamphenicol (50 mg/L), was inoculated with a single colony of E. coli BLR(DE3)pLysS containing
pET-15b-H6-CP or pET-15b-H6-ELP[V4L4G1-9]-CP(ΔN26) and was incubated at 30 °C overnight. This
overnight culture was used to inoculate 900 mL of 2xYT medium supplemented with ampicillin (100 mg/L) and chloroamphenicol (50 mg/L). The culture was grown at 30 °C and protein expression was induced during logarithmic growth (OD600 = 0.4-0.6) by addition of IPTG up to 1 mM. After 5 h of
expression, the cells were harvested by centrifugation (4000 g at 4 °C for 15 min) and the pellet was stored at -20 °C overnight.
After thawing, the cell pellet was resuspended in 15 mL lysis buffer (50 mM NaH2PO4, 10 mM imidazole
and 1300 mM NaCl, pH 8.0) and incubated with lysozyme (1 mg/mL) for 30 min on ice. The cells were then lysed by ultrasonic disruption (5 times 5 s, 100% duty cycle and output control 3, Branson Sonifier 250, Marius Instruments Nieuwegein, the Netherlands). The lysate was centrifuged (13000 RPM at 4 °C for 15 min, Microcentrifuge, Heraeus Pico) to remove the cellular debris. The supernatant was incubated with 1 mL Ni-NTA agarose beads for 1 h at 4 °C. Subsequently, the suspension was loaded onto a column, the flow-through was collected and the column was washed twice with 5 mL wash buffer (50 mM NaH2PO4, 20 mM imidazole and 1300 mM NaCl, pH 8.0). Finally, the CPs were eluted from the
column using 10 times 1 mL elution buffer (50 mM NaH2PO4, 250 mM imidazole and 1300 mM NaCl,
pH 8.0). The purification was analyzed by SDS-PAGE (Figure 5.1a). The fractions containing the CPs were combined and dialyzed against a capsid buffer of pH 7.5 (50 mM Tris, 500 mM NaCl, 10 mM MgCl2, 1 mM EDTA, pH 7.5) to obtain the protein dimers. For storage the proteins were assembled by
dialysis against a pH 5.0 buffer (50 mM NaOAc, 500 mM NaCl, 10 mM MgCl, 1 mM EDTA, pH 5.0). Protein concentrations were determined using a Cary 50 Conc (Varian, Middelburg) UV-VIS spectrophotometer using a quartz cuvette with a path length of 0.3 cm. The protein concentrations were calculated using the theoretical extinction coefficients.[38] The proteins were produced with a yield of 60-
80 mg/L of culture and the purity was verified by SDS-PAGE. ESI-TOF (JEOL AccuTOF): H6-CP
calculated 22506.6 Da, found 22507.4 Da and H6-ELP[V4L4G1-9]-CP(ΔN26) calculated 22253.4 Da,
found 22253.2 Da (Figure 5.1b and c).
Mass spectrometry
Protein mass characterization was performed by electrospray ionization time-of-flight (ESI-TOF) on a JEOL AccuTOF. Deconvoluted mass spectra were obtained using MagTran 1.03b2. Isotopically averaged molecular weights were calculated using the “Protein Calculator v3.3” at http://www.scripps.edu/~cdputnam/protcalc.html.
Designing two self-assembly mechanisms into one viral capsid protein
133
Assembly experiments
For the pH-induced and salt-induced
For
assembly of CPs, dialysis was used to change buffer composition. Therefore, 400 µL of CP-dimer solution (1.0 mg/mL in 50 mM Tris, 500 mM NaCl, 10 mM MgCl2, 1
mM EDTA, pH 7.5) was inserted into a dialysis membrane (Spectra/Por 4 dialysis tubing, 12-14 kDa MWCO, 10 mm flat width) and dialyzed against 100 mL of the desired buffer. After 30 min the buffer solution was refreshed and the solution was incubated for another 30 min. All these steps were performed at room temperature (20 °C).
temperature-induced
For
assembly analyzed by SEC, a solution of CP-dimer (1.0 mg/mL in 50 mM Tris, 500 mM NaCl, 10 mM MgCl, 1 mM EDTA, pH 7.5) was inserted into the dialysis membrane and dialyzed against a pH 7.5 capsid buffer with an increased NaCl concentration (50 mM Tris, 1300 mM NaCl, 10 mM MgCl, 1 mM EDTA, pH 7.5) as described above. Then the sample was incubated at the desired temperature (20 °C or 35 °C) for 15 min prior to injection onto the SEC-column.
temperature-induced
Size exclusion chromatography (SEC)
assembly analyzed by TEM, a solution of CP-dimer (1.0 mg/mL in 50 mM Tris, 500 mM NaCl, 10 mM MgCl, 1 mM EDTA, pH 7.5) was inserted into the dialysis membrane and